Unique modifications of translation elongation factorsEva Greganova*, Michael Altmann and Peter Bu¨tikofer Institute for Biochemistry and Molecular Medicine, University of Berne, Switzer
Trang 1Unique modifications of translation elongation factors
Eva Greganova*, Michael Altmann and Peter Bu¨tikofer
Institute for Biochemistry and Molecular Medicine, University of Berne, Switzerland
Keywords
diphthamide; eEF1A; eEF2; eIF5A;
ethanolamine phosphoglycerol; hypusine;
protein modifcation; translation elongation
Correspondence
P Bu¨tikofer, Institute of Biochemistry and
Molecular Medicine, University of Bern,
Bu¨hlstrasse 28, 3012 Bern, Switzerland
Fax: +41 31 631 3737
Tel: +41 31 631 4113
E-mail: peter.buetikofer@mci.unibe.ch
M Altmann, Institute of Biochemistry and
Molecular Medicine, University of Bern,
Bu¨hlstrasse 28, 3012 Bern, Switzerland
Fax: +41 31 631 3737
Tel: +41 31 631 4127
E-mail: michael.altmann@mci.unibe.ch
*Present address
Swiss Tropical and Public Health Institute
Socinstrasse 57, 4002 Basel, Switzerland
(Received 7 April 2011, revised 12 May
2011, accepted 26 May 2011)
doi:10.1111/j.1742-4658.2011.08199.x
Covalent modifications of proteins often modulate their biological func-tions or change their subcellular location Among the many known protein modifications, three are exceptional in that they only occur on single pro-teins: ethanolamine phosphoglycerol, diphthamide and hypusine Remark-ably, the corresponding proteins carrying these modifications, elongation factor 1A, elongation factor 2 and initiation factor 5A, are all involved in elongation steps of translation For diphthamide and, in part, hypusine, functional essentiality has been demonstrated, whereas no functional role has been reported so far for ethanolamine phosphoglycerol We review the biosynthesis, attachment and physiological roles of these unique protein modifications and discuss common and separate features of the target proteins, which represent essential proteins in all organisms
Introduction
Several hundred protein modifications are known
today, making proteomes far more complex than could
be predicted by the encoding genomes Covalent
modi-fications modulate the biological functions or change
the subcellular location of proteins and affect
interac-tions of proteins with a variety of molecules, such as
nucleic acids, lipids or other proteins [1–3] Particular
modifications are usually present on many proteins
and often proteins carry several modifications at multi-ple amino acid residues [4] The synthesis and attach-ment of protein modifications often involves multiple gene products and sets of metabolites, making these events costly for a cell in terms of substrate and energy requirements On the other hand, modifications may generate additional functions for proteins or allow novel pathways of regulation, providing a cell with
Abbreviations
DHS, deoxyhypusine synthase; DOOH, deoxyhypusine hydroxylase; e(a)EF1A, eukaryotic (archaeal) elongation factor 1A; e(a)EF2, eukaryotic (archaeal) elongation factor 2; e(a)IF5A, eukaryotic (archaeal) initiation factor 5A; EF-G, bacterial elongation factor 2; EF-P, bacterial elongation factor P; EF-Tu, bacterial elongation factor 1A; EPG, ethanolamine phosphoglycerol; PE, phosphatidylethanolamine.
Trang 2extra means to diversify and develop While some
modifications are transient and thus depend on rapid
attachment and removal of molecules from target
pro-teins, others are stable and attached to proteins shortly
after their synthesis or before degradation [4]
Among many protein modifications, three are
excep-tional in that they only occur on single proteins:
etha-nolamine phosphoglycerol (EPG), diphthamide and
hypusine Remarkably, the corresponding proteins
carrying these modifications, eukaryotic elongation
factor 1A (eEF1A), eukaryotic elongation factor 2
(eEF2) and eukaryotic initiation factor 5A (eIF5A)
respectively, are all involved in the elongation steps of
translation
Elongation of polypeptide chains during translation
is a conserved process among prokaryotes and
eukary-otes Single steps of elongation consist of (a) binding of
aminoacyl-tRNAs to the A(minoacyl)-site of the
ribo-some, (b) peptide bond formation with the adjacent
peptide-tRNA at the P(eptidyl)-site and (c)
transloca-tion of the extended peptide-tRNA from the A-site to
the P-site and of the previously loaded tRNA from the
P-site to the E(xit)-site These steps are well conserved
between organisms and the enzymatic involvement of
ribosomal RNA at the transpeptidation center is
nowa-days generally accepted Accordingly, homologs of
most factors involved in elongation can be found across
bacterial, archaeal and eukaryotic genomes
eEF1A, eEF2 and eIF5A are phylogenetically
among the most highly conserved proteins Their
bio-logical roles during elongation of translation are as
fol-lows: eEF1A (called EF-Tu in bacteria and aEF1A in
archaea), one of the most abundant cytosolic proteins,
catalyzes binding of aminoacyl-tRNAs to the A-site of
the ribosome In addition, it has been reported to
par-ticipate in a variety of other functions (so called
moon-lighting functions; see below) In contrast, eEF2 (called
EF-G in bacteria and aEF2 in archaea) is involved in
translocation of the peptide-tRNA complex from the
A- to the P-site, while eIF5A (called EF-P in bacteria
and aIF5A in archaea) directly stimulates protein
elon-gation, yet its precise mode of action on the ribosome
is unclear [5] Bacterial EF-P facilitates the proper
positioning of the initiator-tRNA-methionine complex
at the P-site [6]
Both eEF1A and eEF2 are GTP-binding proteins,
i.e their enzymatic activity requires the hydrolysis of
GTP to GDP Interestingly, GTPases involved in
translation elongation show a remarkable structural
similarity pointing at a common ancestral GTPase
(reviewed by [7]) Its presumed function was to
trans-port aminoacyl-tRNAs to an ancestral
membrane-bound self-folding RNA, which catalyzed peptide bond
formation and constituted the original peptidyltrans-ferase center that evolved later into the corresponding domain of the ribosomal large subunit Co-evolution
of translational GTPases with ribosomal structures may have occurred to allow interaction of GTPases with ribosomal structures by addition of new structural elements [7] In accordance with the concept of co-evo-lution between proteins and RNA structures, elonga-tion (and terminaelonga-tion) factors of translaelonga-tion show a remarkable molecular mimicry between proteins and tRNAs For example, the crystal structure of EF-G from Thermus thermophilus perfectly fits the structure
of the ternary prokaryotic EF-Tu-GDPNP-Phe-tRNAPhecomplex [8] In addition, the crystal structure
of EF-P from Escherichia coli with its post-transla-tional lysine modification resembling the covalently bound amino acid lysine charged to the 3¢ end of a tRNA (see below) mimics the structure of a charged tRNA [9]
The unique modifications attached to eEF1A, eEF2 and eIF5A have been known for decades In addition, their biosynthetic precursors and pathways for produc-tion and attachment to protein have been partially established (see below) Surprisingly, their biological functions have remained elusive despite the fact that EPG, diphthamide and hypusine are attached to essen-tial proteins involved in a highly conserved process, i.e elongation of protein translation, and that species-specific variants of the three proteins have been crys-tallized and their 3D structures solved
The aim of this review is to describe common and separate features of EPG, diphthamide and hypusine attachment to their respective acceptor proteins Inter-estingly, despite the fact that not only the function but also the 3D structures of e(a)EF1A⁄ EF-Tu, e(a)EF2⁄ EF-G and e(a)IF5A ⁄ EF-P proteins have been conserved during evolution (Fig 1), the presence of EPG, diphthamide and hypusine shows striking differ-ences: whereas hypusine (or lysine) attachment to e(a)IF5A⁄ EF-P proteins has been demonstrated in all three domains of life, diphthamide modification has only been found in e(a)EF2 of eukarya and archaea but not in EF-G of bacteria, while EPG has so far only been reported in eEF1A of eukarya (Fig 1)
Eukaryotic elongation factors and their unique modifications
eEF1A and EPG eEF1A represents an essential protein involved in pep-tide chain elongation in all eukaryotic cells It interacts
in its GTP-bound form with an aminoacylated tRNA
Trang 3to mediate binding to the acceptor site of a ribosome
via codon–anticodon interaction Following
ribosome-dependent hydrolysis of GTP, eEF1A dissociates from
the ribosome in its GDP-bound form and interacts
with nucleotide exchange factor eEF1B (called EF-Ts
in bacteria) that replaces GDP by GTP to
reacti-vate eEF1A (reviewed in [10,11]) Crystal structures of
eEF1A in complex with subunits of eEF1B show that
eEF1A from Saccharomyces cerevisiae consists of three
distinct structural domains [12,13] The N-terminal
domain I contains the binding site for guanine nucleo-tides whereas binding of aminoacyl-tRNAs occurs in domain II [12,14–17] In addition, domains I and II share the recognition site for the a-subunit of eEF1B [12,13] In S cerevisiae, domain III has been shown to harbor the binding site for the fungal-specific elonga-tion factor 3 [18,19] Beside its canonical role in pro-tein synthesis, eEF1A has been shown to also bind to cytoskeletal proteins and mediate their interactions [20–22] This function, which has been localized to
Fig 1 3D structure of translation elongation factors The 3D structure of representative examples of e(a)IF5A ⁄ EF-P (top row), e(a)EF2 ⁄ EF-G (middle row) and e(a)EF1A ⁄ EF-Tu (bottom row) proteins is drawn to demonstrate the structural similarity between eukarya, archaea and bac-teria The position of the unique modifications hypusine (Hyp), diphthamide (Dph) and ethanolamine phosphoglycerol (EPG) attached to con-served amino acids (numbered) is indicated by arrows Structures represent eIF5A from Homo sapiens (UniProt, Q6IS14), aIF5A from Sulfolobus acidocaldarius (GenBank, CAA44842) and EF-P from E coli (GenBank, AP_004648), eEF2 from S cerevisiae (UniProt, P32324), aEF2 from H salinarum (UniProt, Q9HM85) and EF-G from T thermophilus (UniProt, Q5SHN5), and eEF1A from Mus musculus (GenBank NP_034236), aEF1A from H salinarum (GenBank, NP_281202) and EF-Tu from E coli (GenBank, YP_001465471), and are drawn using the
PYMOL program [99].
Trang 4domains II and III, seems not to be connected to its
role during polypeptide elongation [21,22] In addition,
eEF1A was reported to be involved in signal
transduc-tion processes [23], nuclear export of proteins [24] and
import of tRNAs into mitochondria [25] Based on the
high conservation of the primary sequence of eEF1A
among eukaryotes (Fig S1) and its highly conserved
role during protein synthesis, it can be speculated that
many interactions with its binding partners are
con-served among other eukaryotic organisms
The activity of eEF1A during peptide synthesis has
been reported to be modulated by post-translational
modifications such as phosphorylation [26,27], lysine
methylation (reviewed in [28,29]) and C-terminal
methyl-esterification [30] The precise role of these
modifications is unclear (reviewed in [31]) In contrast,
no studies have been reported on the role of EPG that
is attached to conserved glutamate residues in eEF1A
of several eukaryotes (Fig S1) Chemical and mass
spectrometric analyses demonstrated that murine [32],
rabbit [33] and carrot [34] eEF1A contain two EPG
modification sites, located in domains II and III In
contrast, although both glutamates are conserved in
eEF1A of the protozoan parasite Trypanosoma brucei
(Fig S1), trypanosome eEF1A is modified only by a
single EPG moiety attached to Glu362 in domain III
[35] (Fig 2A) Amino acid point mutations of the
modification site in T brucei eEF1A were found to
prevent attachment of EPG, even when glutamate was
replaced by aspartate [36], demonstrating that EPG attachment is strictly specific for glutamate Interest-ingly, S cerevisiae represents the only eukaryote so far reported where eEF1A is not modified with EPG [28], although the glutamate residue in domain III is con-served among yeast and other eukaryotes (Fig S1) Amino acid sequence comparisons between eEF1A and EF-Tu show that eukaryotic EPG modification sites are not strictly conserved in bacteria (Figs S2 and S3) For E coli, the lack of EPG modification has been proven experimentally [32] Recent analyses of aEF1A from Halobacterium salinarum and Haloquad-ratum walsbyi showed no evidence for the presence of EPG (E Greganova, R Vitale, A Corcelli, M Heller
& P Bu¨tikofer, unpublished results) suggesting that EPG is absent in archaea
Interestingly, despite the high amino acid sequence identity between eEF1A proteins from different eukary-otes, the residues around the EPG modification sites are less well conserved (Fig S1) suggesting that they may not be essential for EPG attachment [36] Additionally, when expressing eEF1A deletion mutants or chimeric proteins consisting of domain III of T brucei eEF1A fused to soluble reporter proteins, a peptide consisting
of 80 amino acids of domain III of eEF1A was found to
be sufficient for EPG attachment to occur, indicating that EPG attachment is dependent on the three-dimen-sional structure of domain III rather than the sequence
of amino acids around the attachment site [36]
Fig 2 Attachment of EPG to eEF1A (A) Predicted 3D structure of eEF1A from T brucei (TriTrypDB Tb927.10.2100) showing three distinct structural domains (I–III) and the EPG attachment site (Glu362) (B) Proposed pathway for attachment of EPG to eEF1A: PE is attached to Glu362 and subsequently deacylated to EPG.
Trang 5The biosynthetic pathway for EPG attachment has
not been firmly established Although an early study
proposed that binding of free ethanolamine to eEF1A
may represent the first reaction towards a stepwise
assembly of EPG [37], the chemical structure of EPG
(Fig 2B) suggests that the entire EPG moiety may
derive from phosphatidylethanolamine (PE) Studies
using T brucei parasites defective in PE biosynthesis
showed that, indeed, PE is a direct precursor of EPG
in T brucei eEF1A [35] Based on these findings, we
propose a model in which eEF1A is first modified by
PE and then becomes deacylated to EPG (Fig 2B)
If correct, such a model would predict that a
PE-linked eEF1A intermediate might transiently bind to
membranes
Surprisingly, although the covalent attachment of
EPG to eEF1A was described more than 20 years ago,
nothing is known about its biological function
eEF2 and diphthamide
The GTPase eEF2 catalyzes the coordinated
move-ment of peptide-tRNA, unloaded tRNA and mRNA,
and induces conformational changes in the ribosome
(reviewed in [38]) Bacterial EF-G, archaeal aEF2 and
eukaryotic eEF2 clearly show structural and functional
homologies (Fig 1) They all consist of six structural
domains (I–V and G¢; Fig 3A) with the binding
pocket for GDP⁄ GTP being located in domain I [39]
It has been shown that, upon binding of the antifun-gal inhibitor sordarin, yeast eEF2 can undergo dra-matic conformational changes involving rotations of
up to 75 of domains IV–V relative to the amino-ter-minal domains I–II and G¢ through a switch in domain III [40] that may be decisive for its transloca-tion activity eEF2 was reported to be negatively regu-lated by phosphorylation by eEF2-kinase leading to a complete arrest of translation elongation (reviewed in [41])
The unique diphthamide [2-(3-carboxyamido-3-(trim-ethylammonio)propyl)-histidine] modification [42] is conserved from archaea to human but is absent in bac-teria (Figs 1 and S4) Diphthamide serves as cellular target for diphtheria toxin from Corynebacterium diph-theriae (reviewed in [43,44]), exotoxin A from Pseudo-monas aeruginosa [45,46] and cholix toxin from Vibrio cholerae [47,48] These toxins catalyze the trans-fer of ADP-ribose from NAD+ to eEF2-bound diph-thamide resulting in irreversible inactivation of eEF2 and cell death
Enzymatic mono-ADP ribosylation is a phylogeneti-cally ancient mechanism to modulate protein function
in prokaryotes, eukaryotes and viruses [49–51] Exo-toxin A mimics part of the 80S ribosomal structure and interacts with diphthamide-modified eEF2 leading
to its ADP ribosylation [52]
Fig 3 Attachment of diphthamide to eEF2 (A) 3D structure of eEF2 from S cerevisiae (PDB, 2P8Z) showing six distinct structural domains (I–V and G¢) and diphthamide attachment to His699 (B) Pathway for diphthamide synthesis: histidine is modified by a reaction sequence involving five separate enzymes (Dph1–5) to diphthine followed by conversion to diphthamide.
Trang 6The biosynthesis of diphthamide involves the
step-wise addition of different functional groups to the side
chain of a distinct histidine residue in eEF2 (His715 in
mammals and His699 in S cerevisiae) by a
coordi-nated action of the conserved enzymes Dph1–Dph5
and a yet unknown amidase (Fig 3B) [53–58] The
diphthamide modification is located at the tip of
domain IV of eEF2 (Fig 3A) that is supposed to
mimic the tRNA anticodon loop [59] To determine
the amino acid requirements of eEF2 for recognition
by diphthamide biosynthetic enzymes, site-directed
mutagenesis was performed on several residues within
the diphthamide-containing loop (Leu693–Gly703) of
yeast eEF2 Upon replacement of six residues by
ala-nine, mutated eEF2 proteins were lacking the
diphtha-mide moiety [46] Similarly, replacement of Gly717 or
Gly719 in mammalian eEF2 led to diphtheria
toxin-resistant cells [60,61]
Despite the fact that this modification was first
described more than 30 years ago [42], its role in normal
cellular function has remained largely elusive
System-atic mutagenesis of yeast eEF2-His699 showed that the
resulting eEF2 proteins were lacking diphthamide and,
consequently, were not ADP-ribosylated by diphtheria
toxin [62] Interestingly, the various yeast eEF2 mutants
were either lethal indicating a key role of His699 for
eEF2 function or led to temperature-sensitive growth of
yeast indicating that diphthamide attachment to eEF2 is
not strictly required for cell growth [62,63] The
dispens-ability of diphthamide for eEF2 function was later
confirmed by mutagenesis of eEF2-His715 in mammals
[64] Moreover, yeast mutants lacking Dph1, Dph2,
Dph4 or Dph5 genes showed no growth phenotypes
compared with wild-type cells [58]
The non-essentiality of diphthamide and the Dph
enzymes raises the question why such a complex
post-translational modification has been maintained in
archaea and eukarya It has been postulated that
essential functions of diphthamide may only become
apparent under certain circumstances, e.g in the
text of a multi-cellular organism or during stress
con-ditions [65] In mouse and human, Dph1 has been
identified as a tumor suppressor gene [66–68] In mice,
knockout of one Dph1 allele lead to increased tumor
development whereas loss of both Dph1 alleles resulted
in death at an early age [69] Similarly, Dph3 knockout
mice showed embryonic lethality [70] These
observa-tions indicate a potential role for diphthamide in the
control of tumorigenesis, cell growth and embryonic
development However, the effects caused by loss of
dph genes in mammals may be related to other
func-tions of the gene products such as tRNA modification
by Dph3 [71]
As mentioned, the importance of diphthamide in eEF2 function may become apparent during stress con-ditions [65] For instance, yeast strains expressing H699N eEF2 or lacking Dph2 or Dph5 are viable but reveal increased frequency in ())1 ribosomal frame shifting [59] Furthermore, diphthamide has been pro-posed to protect ribosomes from ribosome-inactivating proteins by showing that cultured Chinese hamster ovary cells lacking the diphthamide biosynthetic enzymes Dph2, Dph3 or Dph5 were threefold more sensitive towards ricin than wild-type cells [65] After complementation with the corresponding dph genes, the mutant cells gained resistance to ricin
Alternatively, diphthamide may serve as a regulatory modification site of eEF2 It has been previously pos-tulated that ADP ribosylation by diphtheria toxin may represent a normal cellular control mechanism (reviewed in [72]) In mammalian cells, an endogenous ADP-ribosyltransferase activity specific for eEF2 has been described [73–75] that may function in controlling protein synthesis
eIF5A and hypusine For many years, eIF5A was assumed to be involved in translation initiation [76–78] Only recently, studies in yeast demonstrated that eIF5A promotes translation elongation rather than translation initiation [5,14,79] eIF5A stimulates translation directly and functions as
a general translation elongation factor in a manner determined by its hypusine modification [5]
The unique hypusine [Ne-(4-amino-2-hydroxybutyl)-lysine] modification [80] attached to domain I of eIF5A has been found in all eukaryotes examined so far (reviewed in [81,82]) (Fig 4A) In addition, it also occurs in certain archaea [83] but has not been detected in bacteria However, in E coli the conserved lysine residue in domain I of EF-P (Fig S5) is modi-fied by lysine by a paralog of lysyl-tRNA synthetase Interestingly, the structure of EF-P mimics that of L-shaped tRNA and its lysylation site (Lys34) corre-sponds to the tRNA 3¢ end [9] Domains I and II are highly conserved among all organisms; however, eIF5A and aIF5A lack a carboxyterminal domain III found in bacterial EF-P (see Fig 1) While the amino-terminal domain I is located close to the aminoacyl acceptor stem of initiator tRNA bound to the P-site of the 70S ribosome, the carboxyterminal domain III of bacterial EF-P is positioned close to the anticodon stem-loop [6]
Hypusine is formed by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOOH) (Fig 4B)
Trang 7Both enzymes are highly conserved among eukaryotes
and display similar structural requirements for their
substrates, eIF5A-lysine and eIF5A-deoxyhypusine
[84–86] While neither DHS nor DOOH are found in
bacteria, a gene homolog for DHS has been identified
in archaea However, it is not clear how hypusinated
aIF5A is generated in archaea [87] Mutations at the
hypusine attachment site Lys50 in human eIF5A
(Fig 4A) completely blocked deoxyhypusine synthesis
whereas substitutions in its vicinity resulted in reduced
efficiency of deoxyhypusine synthesis or inhibition of
the hydroxylation reaction catalyzed by DOOH [88]
A truncated peptide consisting of 80 residues of human
eIF5A (amino acids 10–90; expressed in E coli) was
nearly as good a substrate as the full-length protein
for hypusine attachment [85,86]
Disruption of the eIF5A [89,90] or DHS [91,92] gene
results in a lethal phenotype In contrast, the DOOH
gene does not appear to be essential in S cerevisiae
since growth of a DOOH null mutant strain was only
slightly reduced compared with the parental strain [93]
However, in multi-cellular organisms such as
Caenor-habditis elegans or Drosophila melanogaster
inactiva-tion of the DOOH gene was found to be recessively
lethal [94,95] Thus, although in single cell eukaryotes
deoxyhypusinated eIF5A is sufficient to perform its
essential cellular functions, multi-cellular eukaryotes require hypusinated eIF5A In addition to the above-mentioned phenotypes, hypusine is necessary for homodimerization of eIF5A and affects its subcellular localization [96,97] However, the precise mode of eIF5A action and how hypusine modulates eIF5A function remain to be answered It is possible that eIF5A fulfills the same function as its bacterial ortho-log EF-P, which has been shown to catalyze the forma-tion of the first peptide bond in protein synthesis (reviewed in [98]) The recent resolution of its crystal structure [6] has provided new insights into the function
of EF-P, indicating that it allows proper positioning of initiator met-tRNA at the P-site of the ribosome in a situation where the E-site of the ribosome is not occu-pied by unloaded tRNA
Conclusions
We have reviewed the unusual post-translational modi-fications of three different translation elongation fac-tors that are present in all cells and participate in a conserved mechanistic pathway among eukaryotes and prokaryotes Though not essential in all organisms (Fig 1), EPG, diphthamide and hypusine are impor-tant to maintain the activity (and probably also the
Fig 4 Attachment of hypusine to eIF5A (A) Predicted 3D structure of human eIF5A (PDB, 1FH4) showing two distinct structural domains (I, II) and the hypusine attachment site (Lys50) (B) Pathway for hypusine synthesis: spermidine is attached to lysine and subsequently modi-fied to hypusine.
Trang 8proper structure) of acceptor proteins The biological
significance of these modifications may only become
evident in vivo or under certain stress or competition
conditions, which so far have not been mimicked in
the laboratory
In all eukaryotes studied, the function of eIF1A,
eEF2 and eIF5A is essential for cell survival To our
knowledge, cross-complementation experiments with
paralog prokaryotic and eukaryotic factors have so far
not been reported One possible reason why such
experiments may not work would be due to
co-evolu-tion of these proteins with their interacting partners
which might have given rise to subtle differences that
do not allow for cross-complementation of single
para-logs in different organisms
Whether EPG, diphthamide and hypusine play a
role in protein–protein interactions is unknown The
availability of efficient knockout⁄ knockin and
knock-down techniques using mono- and multi-cellular
organisms may allow our knowledge about the
impor-tance of these modifications to be extended in the near
future
Why are the three modifications EPG, diphthamide
and hypusine restricted to single proteins and why
are the three modified proteins all involved in
elonga-tion of translaelonga-tion? We propose that the
modifica-tions are remnants of an evolutionary process that
might have been more common in an ancient world,
i.e that multiple proteins were modified by EPG,
diphthamide and hypusine During the course of
evo-lution, however, these modifications may have mostly
disappeared, except for the translation elongation
proteins e(a)EF1A⁄ EF-Tu, e(a)EF2⁄ EF-G and
e(a)IF5A⁄ EF-P, which are highly conserved between
organisms and for which EPG, diphthamide and
hypusine may fulfill important functions to enhance
accuracy or catalytic activity of enzymes interacting
with translating ribosomes For diphthamide, and in
part hypusine, functional essentiality has been
demon-strated In contrast, no functional role has so far
been reported for EPG
Acknowledgements
We thank U Baumann (University of Ko¨ln) and
G Hernandez (McGill University, Montreal) for advice
during preparation of the manuscript E.G thanks
P Ma¨ser (Swiss Tropical and Public Health Institute,
Basel) for support P.B thanks G Moore for
stimula-tion and input and O Bu¨tikofer for support Research
in our laboratories is supported by Swiss National
Science Foundation grants 31003A-130815 to P.B and
31003A-119996 to M.A
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